Abstract
Iron (Fe) is an essential micronutrient for humans. Fe deficiency disease is widespread and has led to extensive studies on the mechanisms of Fe uptake and storage, especially in staple food crops such as rice. However, studies of functionally related genes in rice and other crops are often time and space demanding. Here, we demonstrate that transgenic Arabidopsis suspension culture cells and Arabidopsis plants can be used as an efficient expression system for gain-of-function study of selected transporters, using Fe transporters as a proof-of-principle. The vacuolar membrane transporters OsVIT1 and OsVIT2 have been described to be important for iron sequestration, and disruption of these two genes leads to Fe accumulation in rice seeds. In this study, we have taken advantage of the fluorescent-tagged protein GFP-OsVIT1, which functionally complements the Fe hypersensitivity of ccc1 yeast mutant, to generate transgenic Arabidopsis suspension cell lines and plants. GFP-OsVIT1 was shown to localize on the vacuolar membrane using confocal microscopy and immunogold EM. More importantly, the Fe concentration, as well as the concentration of Zn, in the transgenic cell lines and plants were significantly increased compared to that in the WT. Taken together, our study shows that the heterologous expression of rice vacuolar membrane transporter OsVIT1 in Arabidopsis system is functional and effectively enhances iron accumulation, indicating an useful approach for studying other putative transporters of crop plants in this system.
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Acknowledgments
This work was supported by grants from the Research Grants Council of Hong Kong (CUHK4666011, 46112, 466613, CUHK2/CRF/11G, C4011-14R, and AoE/M-05/12), NSFC/RGC (N_CUHK406/12), NSFC (31270226 and 31470294), and Shenzhen Peacock Project (KQTD201101) to L. Jiang. This work was also supported in part by funding from the US Department of Agriculture, Agricultural Research Service through Cooperative Agreement Number 58-6250-0-008 to M.A. Grusak. The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.
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Supplement Figure S1
The concentration of macronutrients Ca, Mg and P in Arabidopsis cell lines (PDF 532 kb)
Supplement Figure S2
The concentration of Fe (A), Mn (B), Zn (C) and Cu (D) in Arabidopsis stem tissues (PDF 1529 kb)
Supplement Figure S3
The concentration of macronutrients Ca, Mg and P in Arabidopsis stem (A) and silique (B) tissues (PDF 1123 kb)
Supplement Figure S4
. The concentration of Fe (A), Mn (B) and Zn (C) in Arabidopsis stem tissue when supplemented with 50 μM Fe-EDTA (PDF 736 kb)
Supplement Figure S5
The concentration of Ca, Mg, P and Cu in Arabidopsis stem (A) and silique (B) tissues when supplemented with 50 μM Fe-EDTA (PDF 1.39 MB)
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Wang, X., Zhong, F., Woo, C.H. et al. A rapid and efficient method to study the function of crop plant transporters in Arabidopsis . Protoplasma 254, 737–747 (2017). https://doi.org/10.1007/s00709-016-0987-6
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DOI: https://doi.org/10.1007/s00709-016-0987-6